Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3015—Breast
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S424/00—Drug, bio-affecting and body treating compositions
  • Y10S424/804—Drug, bio-affecting and body treating compositions involving IgG3, IgG4, IgA, or IgY
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/948—Microorganisms using viruses or cell lines
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/808—Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/861—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgG3, IgG4, IgA, or IgY
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/863—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgM
  • Y10S530/864—Monoclonal

Definitions

• This invention is in the fields of immunology and cancer diagnosis and therapy. More particularly it concerns murine monoclonal anti-human breast cancer antibodies, hybridomas that produce these antibodies, immunochemicals made from those antibodies, and diagnostic and therapeutic methods that use those immunochemicals.
• mice were immunized and boosted with human milk fat globule proteins, breast cancer cell lines or breast cancer membrane extracts. Immune splenocytes were fused with mouse myeloma cells and hybridomas were selected based on some specificity of the culture media for breast cancer or breast cancer antigens.
• the normal tissue reactivities of these prior antibodies are different than the normal tissue reactivities of the antibodies of the present invention.
• a principal aspect of the invention concerns murine monoclonal antibodies that:
• Preferred embodiments of these antibodies are those designated 260F9, 113F1, 2G3, 280Dll, 266B2, 33F8, 245E7, 454Cll, 317G5, 520C9, 369F10, and functional equivalents thereof.
• the murine x murine hybridomas that produce the above described antibodies and progeny of those hybridomas are other aspects of the invention.
• the invention also includes a method of preparing a hybridoma as defined above comprising fusing murine tumor cells with murine splenocytes obtained from a mouse immunized with human breast cancer immunogen and selecting for hybridomas producing antibody as defined above.
• a further aspect of the invention is a method of producing antibody as defined above comprising culturing a hybridoma having the ability to produce such antibody, optionally a hybridoma which has been prepared by effecting a method as claimed above.
• Another aspect of the invention relates to immunotoxins and their preparation by conjugating
• Another aspect of the invention concerns labeled derivatives of the above described monoclonal antibodies.
• Another aspect of the invention concerns a method of killing human breast cancer cells by contacting the cells with a cytocidally effective amount of one or more of the above described immunotoxins.
• kits for determining whether a human cell is a breast cancer cell involve incubating the cells with the monoclonal antibodies or labeled derivatives thereof. When the labeled derivatives are used, the presence of labeled binary immune complexes on the cells is read directly. When unlabeled antibody is used the cells are further incubated with a labeled antibody against the monoclonal antibody and the presence of labeled ternary immune complexes on the cells is read.
• the term "monoclonal antibody” means an antibody composition having a homogeneous antibody population. It is not intended to be limited as regards the source of the antibody or the manner in which it is made.
• the term "functional equivalent” means a monoclonal antibody that: (a) crossblocks an exemplified monoclonal antibody; (b) binds selectively to human breast cancer cells; (c) has a G or M isotype; (d) binds to the same antigen as determined by immunoprecipitation or sandwich immunoassay; and (e) when conjugated to ricin A chain, exhibits a TCID 50% against at least one of MCF-7, CAMA-1, SKBR-3, or BT-20 cells of less than about 10 nM.
• progeny is intended to include all derivatives, issue, and offspring of the parent hybridoma that produce the monoclonal anti-human breast cancer antibody produced by the parent, regardless of generation or karyotypic identity.
• the antibody-producing fusion partners that are used to make the hybridomas of this invention are generated by immunizing mice with live human breast cancer cells or membrane extracts made therefrom.
• the mice are inoculated intraperitoneally with an immunogenic amount of the cells or extract and then boosted with similar amounts of the immunogen.
• Spleens are collected from the immunized mice a few days after the final boost and a cell suspension is prepared therefrom for use in the fusion.
• Hybridomas are prepared from the splenocytes and a murine tumor partner using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature (1975) 256:495-497 as modified by Buck, D. W., et al, In Vitro (1982) 18:377-381.
• Available murine myeloma lines such as those from the Salk Institute, Cell Distribution Center, San D iego, California, USA, may be used in the hybridization.
• the technique involves fusing the tumor cells and splenocytes using a fusogen such as polyethylene glycol.
• the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells.
• a selective growth medium such as HAT medium
• the hybridomas are expanded, if desired, and supernatants are assayed for anti-human breast cancer activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay) using the immunizing agent (breast cancer cells or membrane extract) as antigen.
• immunoassay procedures e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay
• Positive clones are characterized further to determine whether they meet the criteria of the invention antibodies.
• Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures.
• the monoclonal antibodies may be isolated from the culture media or body fluids, as the case may be, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
• the important characteristics of the monoclonal antibodies are (1) their immunoglobulin class, (2) their selectivity for human breast cancer cells and the range of human breast cancer cells to which they bind and (3) their usefulness in making effective anti-human breast cancer immunotoxins.
• the selectivity and range of a given antibody is determined by testing it against panels of (1) human breast cancer tissues and cells and (2) normal human tissues or cells of breast or other origin.
• approximately twenty-two thousand growing hybridoma cultures were initially screened against the immunizing breast tumor membranes or cell line, a panel of eight normal tissue membranes, a fibroblast cell line and a breast tumor frozen section. Clones that reacted with the neoplastic materials but not the normal materials were identified in this initial screen and chosen for isotyping and additional screening for selectivity and range.
• the additional screening involved: sixteen normal tissue sections, five normal blood cell types, eleven nonbreast neoplasm sections, twenty-one breast cancer sections and fourteen breast cancer cell lines. Antibodies were deemed to bind selectively to breast cancer if they bound strongly to less than about 1/3 of the normal tissues and blood cell types. One hundred twenty-seven antibodies were purified and tested on the additional screen.
• Antibodies exhibiting acceptable selectivity and range were conjugated to ricin A chain using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or iminothiolane (IT) as a coupling agent.
• SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
• IT iminothiolane
• the conjugates were tested against MCF-7, CAMA-1, SKBR-3, and BT-20 cells in a 24-hour tissue culture assay.
• Sixteen of the antibodies exhibited acceptable immunotoxin activity (TCID 50% of less than 10 nM) against at least one of these breast tumor lines. Seven of the sixteen were found to recognize the same 210,000 dalton antigen, with six of the seven probably recognizing the same epitope but differing in affinity.
• immunotoxins conjugates of the antibody and a cytotoxic moiety
• labeled e.g., radiolabeled, enzyme-labeled, or fluorochrome-labeled
• the cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment ("A chain") of such a toxin.
• Enzymatically active toxins and fragments thereof are preferred and are exemplified by dipntheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPi, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin.
• Ricin A chain, nonbinding active fragments of dipntheria toxin, abrin A chain, and PAPII are preferred.
• Conjugates of the monoclonal antibody and such cytotoxic moieties may be made using a variety of bifunctional protein coupling agents.
• reagents examples include SPDP, IT, bifunctional derivatives of imidoesters such as dimethyl adipimidate HC1, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis- active fluorine compounds such a 1,5-difluoro-2,4-dinitrobenzene.
• imidoesters such as dimethyl adipimidate HC1
• active esters such as disuccinimidyl suberate
• aldehydes such as glutaraldehyde
• bis-azido compounds such as bis(p-azidobenzoyl) hexan
• the conjugates When used to kill human breast cancer cells in vitro for diagnostic purposes, the conjugates will typically be added to the cell culture medium at a concentration of at least about 10 nM.
• the formulation and mode of administration for in vitro use are not critical. Aqueous formulations that are compatible with the culture or perfusion medium will normally be used. Cytotoxicity may be read by conventional techniques to determine the presence or degree of breast cancer.
• the immunotoxins When used in vivo for therapy, the immunotoxins are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's tumor burden). They will normally be administered parenterally, preferably intravenously.
• the dose and dosage regimen will depend upon the nature of the cancer (primary or metastatic) and its population, the characteristics of the particular immunotoxin, e.g., its therapeutic index, the patient, and the patient's history.
• the amount of immunotoxin administered will typically be in the range of about 0.1 to about 10 mg/kg of patient weight.
• the immunotoxins will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
• a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers.
• the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
• the immunotoxin will typically be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
• Cytotoxic radiopharmaceuticals for treating breast cancer may be made by conjugating high linear energy transfer (LET) emitting isotopes (e.g., Y, Pr) to the antibodies.
• LET linear energy transfer
• cytotoxic moiety as used herein is intended to include such isotopes.
• the labels that are used in making labeled versions of the antibodies include moieties that may be detected directly, such as fluorochromes and radiolabels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
• moieties such as 32p , 125 I , 3 E, 14 C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase.
• the antibodies may be tagged with such labels by known methods.
• coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bis-diazotized benzadine and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels.
• the antibodies and labeled antibodies may be used in a variety of immunoimaging or immunoassay procedures to detect the presence of breast cancer in a patient or monitor the status of such cancer in a patient already diagnosed to have it. When used to monitor the status of a cancer a quantitative immunoassay procedure must be used. In such monitoring assays are carried out periodically and the results compared to determine whether the patient's tumor burden has increased or decreased.
• Common assay techniques that may be used include direct and indirect assays. Direct assays involve incubating a tissue sample or cells from the patient with a labeled antibody. If the sample includes breast cancer cells, the labeled antibody will bind to those cells.
• tissue sample After washing the tissue or cells to remove unbound labeled antibody, the tissue sample is read for the presence of labeled immune complexes.
• tissue or cell sample is incubated with unlabeled monoclonal antibody.
• the sample is then treated with a labeled antibody against the monoclonal antibody (e.g., a labeled antimurine antibody), washed, and read for the presence of labeled ternary complexes.
• a labeled antibody against the monoclonal antibody e.g., a labeled antimurine antibody
• kits will typically comprise: the antibody in labeled or unlabeled form in suitable containers, reagents for the incubations and washings, a labeled antimurine antibody if the kit is for an indirect assay, and substrates or derivatizing agents depending on the nature of the label.
• Human breast cancer antigen controls and instructions may also be included.
• Human breast cancer cell lines were obtained from the Breast Cancer Task Force, the American Type Culture Collection (ATCC), and from Dr. Jorgen Fogh at Memorial Sloan Kettering. The cells were maintained and passaged as recommended by the Breast Cancer Task Force, the ATCC and Dr. Fogh.
• For immunizations either membrane extract containing 100 u g of protein (Lowry assay) or ten million live breast cancer cells were inoculated intraperitoneally into five week old Balb/c mice. The mice were boosted identically twice at monthly intervals. Three days after the last boost, the spleens were removed for cell fusion.
• Somatic cell hybrids were prepared by the method of Buck, D. W., et al, supra, using the murine myeloma Tin Sp-2/0/Agl4. All hybridoma cell lines were cloned by limiting dilution. Half of the fusions employed splenocytes from mice immunized with breast cancer membrane extracts and half used splenocytes from mice immunized with live breast cancer cell lines. Eighty-three thousand four hundred twenty-four wells were generated from those fusions, of which 22,459 exhibited hybridoma growth.
• Hybridoma supernatant was assayed for reactive antibody in either a sold phase enzyme-linked immunosorbent assay (ELISA) with the immunizing breast cancer membrane extract or an indirect immunofluorescence assay with the immunizing breast cancer cell line.
• ELISA enzyme-linked immunosorbent assay
• 40 ⁇ l of 0.1 mg/ml breast cancer membrane protein were placed in polyvinyl chloride (PVC) microtiter wells for 12 hr at 4°C. The extract was aspirated and the wells washed with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). The wells were then incubated with 45 ⁇ 1 of a 1:10 dilution of hybridoma supernatant.
• PBS phosphate buffered saline
• BSA bovine serum albumin
• the diluent was media with 25 mM of a buffer, 10% bovine serum, and 0.1% sodium azide. After 30 min at room temperature, the wells were again washed and incubated 45 min at 37°C with a 1:200 dilution of peroxidase conjugated goat anti-mouse IgG. The diluent was PBS. The wells were then washed with PBS and reacted with 200 ⁇ 1 of 2,2-azino-di(3-ethylbenzthiazoline sulphonic acid) in 0.1 M sodium citrate buffer pH 4.2 for 30 min at room temperature. Optical density was measured at 405 nm on a MicroElisa Reader.
• the indirect immunofluorescence cell line assay we placed one hundred thousand breast cancer cells of the immunizing cell line overnight with appropriate media in each chamber of a set of eight chambered slides. Similarly, one hundred thousand fibroblast cells from cell line CC95 were incubated overnight in chambered slide wells. The cells were washed with PBS containing 1% BSA. The wells, both breast cancer and fibroblast, were incubated for 30 min at 4°C with 1:10 dilutions of hybridoma supernatant.
• the cells were again washed and incubated 30 min at 4°C with a 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated goat F(ab') 2 anti-mouse Ig.
• FITC fluorescein isothiocyanate
• the cells were washed three times, fixed in 1.5% formaldehyde in PBS for five min, chambers removed and rinsed in PBS.
• the slides were then mounted in a composition containing polyvinyl alcohol, glycerol, biffers and a preservative and examined with a fluorescence microscope.
• Hybridoma wells showing strong fluorescent binding to breast cancer cells but no fluorescent binding to fibroblasts were saved. Five thousand one hundered fifty-six hybridoma wells revealed breast cancer reactivity in the initial screen.
• the 1101 hybridoma supernatants were tested on frozen sections of human breast carcinoma tissues. Six micron sections were attached to slides, fixed 10 min in acetone at 4°C, dried 10 min at room temperature, washed with PBS, blocked with horse serum and incubated 20 min at room temperature with 200 ⁇ 1 neat hybridoma supernatant.
• the slides were washed with PBS, and finally incubated 20 min at 37°C with a 1:50 dilution of peroxidase conjugated rabbit anti-mouse Ig, washed again with PBS, and finally incubatged 7.5 min at 37°C with 0.5 mg/ml diaminobenzidine in 0.05 M Tris buffer pH 7.2 containing 0.01% hyudrogen peroxide.
• the slides were stained with hematoxylin, dehydrated and mounted in a medium containing 35.9% methyl/n-butylmethacrylate copolymer, 7.1% butyl benzyl phthalate, and 0.3% 2,6-ditertbutyl-p-cresol.
• One hundred twenty-four wells yielded breast cancer selective binding and were cloned.
• Immunoglobulin class and subclass of the monoclonal breast cancer selective antibodies were determined. Antibodies were also internally labeled by growing 2-3 x 10 6 hybridoma cells for 4 hr in methionine-free medium containing 0.2 ⁇ Ci 35 S methionine. 35 S-labeled antibodies were immunoprecipitated with fixed staphylococcus A cells, or with the composition used to fix the staphylococcus A cells precoated with rabbit anti-mouse immunoglobulin, and the immunoprecipitates were analyzed by SDS-PAGE to determine antibody light and heavy chain mobility, lack of extra chains, and the ability of each antibody to bind staphylococcal protein A.
• mice were primed with 0.5 ml pristane intraperitoneally (ip) and after 10-14 days inoculated with one million log phase hybrodima cells in PBS. Ascites fluid was stored at -70°C and thawed and filtered through a 0.8 micron filter unit before further purification.
• IgG antibodies that bound staphylococcal protein A were purified by affinity chromatography on protein A-chromatographic resin containing agarose, dextron and/or acrylamide with pH step gradient elution. IgG antibodies that did not bind protein A were precipitated by addition of ammonium sulfate to 40% saturation at 0°C. The precipitates were redissolved in PBS, dialysed to 20 mM Tris pH 7.2 and chromatographed on a 1.6 x 50 cm column of diethylaminoethyl cellulose (DEAE) eluting with a 1.5 liter 0-600 mM NaCl gradient at 4°C at a flow rate of 1 ml/min. In each case, column fractions were monitored by SDS-PAGE and the purest antibody fractions were pooled, concentrated to 1-3 mg/ml, dialysed to PBS/0.02% NaN 3 , and stored at 4°C.
• DAE diethylaminoethyl cellulose
• IgM antibodies were purified by gel filtration on a 2.6 x 40 cm column of chromatographic resin containing agarose, dextron and/or acrylamide eluting with PBS/0.01% sodiumn azide at room temperature at a flow rate of 1 ml/min.
• the purified antibodies were tested by immunoperoxidase section staining on sections of sixteen normal tissues, and by immunofluorescent cell sorting on five blood cell types. Immunoperoxidase staining was performed as above except that known dilutions of purified antibodies in PBS in the range of 1-40 ⁇ g/m] were used instead of hybridoma supernatants. The pure antibodies were first titrated to find the minimal concentration giving strong immunoperoxidase staining on breast cancer sections and then used at that concentration for the normal tissue tests.
• Peripheral blood cells (platelets, lymphocytes, red bood cells, granulocytes, and monocytes) were prepared by centrifugation using a medium which separates monocytes from polymorphonuclear leucocytes.
• the cells were reacted with antibody at the optimal concentration determined above for 30 min at 4°C, washed, reacted with a 1:50 dilution of fluorescence isothiocyanate-conjugated goat anti-mouse Ig for 30 min at 4°C, washed again and examined in a cell sorter.
• the wash buffer and diluents were PBS with 1% gelatin and 0.02% sodium azide.
• the cell sorter was equipped with a 76 micron nozzle and a one watt argon ion laser at 488 nm.
• An 80 mm confocal lens was used on the optical rail assembly for focusing.
• Other filters used were a 515 nm interference filter and a 515 nm absorbance filter (for scattered laser light) and a neutral density 1.5 filter for forward angle light scatter. Contour plots of log fluorescein fluorescence versus forward angle light scatter were used for sample analysis.
• the breast cancer selective antibodies were tested by immunoperoxidase staining on frozen sections of 27 different breast tumors.
• the breast cancers used for section staining were all infiltrating intraductal carcinomas, so no correlation of antibody binding with histologic type of breast cancer could be made.
• no correlation between antibody binding and the nodal status or estrogen receptor status was found for the twelve tumors for which donor information was available.
• Antibodies reacted equally well with metastatic and primary breast tumors. The results of these tests for the claimed antibodies are reported in Table 2 below.
• Antibodies were further evaluated for range of breast cancer recognition by cell line immunofluorescence assays on 14 breast cancer cell lines. Table 3 below reports the results of these tests for the claimed antibodies.
• the claimed antibodies were conjugated to ricin toxin A chain (RTA) treated with SPDP as described by Carlsson, J., et al, Biochem J (1978) 173:723-737 or with iminothiolane (IT).
• RTA ricin toxin A chain
• SPDP ricin toxin A chain
• I iminothiolane
• SPDP (20 mM in ethanol) was added in a 20-fold molar excess to antibody and following a 30 min incubation at room temperature, the unreacted SPDP was removed by dialysis against PBS. The extent of derivatization was determined by measuring the release of pyridine-2-thione at 343 nm after reduction with dithiothreitol (DTT). Depending on the antibody, three to eight lysine amino acid groups (per antibody molecule) were converted to the pyridyl-disulfide derivative.
• DTT dithiothreitol
• the SPDP-treated antibodies were conjugated with RTA.
• the RTA was reduced with 50 mM DTT, then desalted on a column of chromatographic resin containing agarose, dextran and/or acrylamide to remove DTT from protein.
• Reduced RTA was added in a three- to five-fold molar excess over pyhridyl-disulfide antibody.
• a typical reaction mixture (1 ml) consisted of 7 ⁇ M antibody and 30 pm RTA. The reaction was allowed to proceed overnight at 4°C.
• the extent of conjugation of RTA to antibody was determined spectrophotometrically by measuring the release of pyridine-2-thione. On the average, conjugates contained two to three RTA molecules per antibody molecule. This was confirmed by nonreducing SDS-PAGE gels (7.5%), which also revealed that the typical conjugate preparation contained 10%-30% free antibody.
• Tne conjugate mixture was chromatographed on an HPLC size exclusion column to separate conjuates from residual unreacted RTA.
• the column was equilibrated in 0.1 sodium sulfate/0.02 M sodium phosphate pH 6.8. Conjugate mixture (0.7 ml) was injected, then chromatographed at a flow rate of 1 ml/min (room temperature). Fractions of 0.5 ml were collected and the peak conjugate fractions were pooled and filter sterilized prior to cytotoxicity testing.
• P-EDTA buffer Approximately 30 mg/ml antibody in 0.10 M Na phosphate, 0.001 M Na EDTA, pH 8.0 (hereafter referred to as P-EDTA buffer) is reacted with 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at room temperature for about 15 min and then chilled to 0°C in an ice bath. Enough IT is added to this solution to give 2.5 IT molecules/antibody molecule, and the resulting solution is stored at 0-5°C against three 100-fold excess volumes of P-EDTA buffer.
• DTNB 5,5'-dithiobis-(2-nitrobenzoic acid)
• RTA normally stored in P-EDTA containing 1 mM DTT, is ultrafiltered to a concentration between 10 and 15 mg/ml and dialyzed at 0-5°C against three 100-fold excess volumes of P-EDTA. Enough RTA is added to the derivatized antibody to give 1.0-1.2 free thiols on RTA/blocked thiol on derivatized antibody. This mixture is incubated at room temperature for 2 hr.
• the coupling reaction mixture is applied to a column of a chromatographic resin based on a blue dye hooked up to a solid support, which mixture is then eluted with P-EDTA at room temperature.
• the column is scaled to contain approximately 2 ml of bed volume per mg of starting antibody.
• the eluant is switched to P-EDTA containing 1 M NaCl.
• Immunoconjugate and unreacted RTA are eluted in this buffer as a very sharp peak, which is pooled and dialyzed at 0-5°C against one 10-fold excess volume of 0.15 M Na phosphate, pH 7.1 (hereafter referred to as p i buffer).
• the dialyzed protein is applied to a column of a get at 0-5°C and eluted with buffer at a flow rate of 6 cm/hr.
• the column is scaled to contain at least 25 ml of bed volume/ml of applied protein.
• Immunoconjugate is eluted as a single peak, slightly after the excluded volume, baseline- resolved from following peaks of dimerized and monomeric RTA. TGhe pooled immunoconjujgate peak is ultrafiltered at 35 psi to a final concentration of 5.0 mg/ml and filter-sterilized.
• test human breast cancer lines used in the cytotoxicity tests were MCF-7, CAMA-1, SKBR-3, and BT-20.
• human fibroblast cell lines CC95 and WI-38 were used as negative controls.
• test cells in 1 ml medium were added to a set of 8 ml glass vials, followed by the addition of conjugate dilutions (in PBS containing 100 ⁇ g/ml BSA). After incubation at 37°C for 22 hr, the medium was aspirated, the monolayers were washed with PBS, and methionine-free medium supplemented with 35 S methionine was added. The vials were further incubated 2 hr at 37°C, the medium was removed, and the cells were washed twice with 2 ml of 10% trichloroacetic acid containing 1 mg/ml methionine.
• Conjugates of 245E7, 280D11, and 260F9 with RTA were made as above using iminothiolane or SPDP as a coupling agent.
• the efficacies of these conjugates against MX-1 human breast tumor cells in vivo was evaluated as follows.
• mice Female athymic Balb/c-nu/nu mice (20-24 g) were used. Fragments, 1.0 mm 3 , were obtained from 600-800 mm 3 tumors with no signs of central necrosis and packed into a syringe. Mice were implanted s.c. with 0.05 ml of the suspension in the axillary region with mediolateral puncture. On day 7 or 14 after implant the mice were weighed and their tumor burdens were evaluated by measuring the implants with calipers. Mice were grouped according to mean tumor size.
• the conjugates were injected i.v. into the tail vein of control mice Q2 D x 6 to determine the maximum tolerable dose of the particular conjugate. Based on these results, dose regimens for aaministering the conjugates to tumor-bearing mice were selected. Groups of tumor-bearing mice and control mice were injected i.v. with the conjugates according to the chosen regimens. Animal reactions, side effects, and mortalities were monitored daily along with tumor volume and animal weight measurements. Changes in tumor volume at the end of the test period were calculated based on the average of the sum of measurements over the test period. The results of these tests are reported in Table 6 below.
• the antibodies were iodinated and tested for binding to MCF-7 or BT-20 cells.
• the antibodies were labeled with 125 I using chloramine T to a specific activity of approximately 10 ⁇ Ci/ug.
• 100,000 cpm of two of the labeled antibodies in 0.5 ml fetal calf serum was serially absorbed with five aliquots of target cells for 15 min at 0°C (generally 4,000,000 MCF-7 breast cancer cells per aliquot), and the remaining radioactivity in the supernatant after each absorption was determined.
• association constants For measurements of association constants, known concentrations of labeled and unlabeled monoclonal antibodies were incubated with target cells in fetal calf serum for 15 min in ice. Aliquots of the cell/antibody mix were then counted in a gamma counter or filtered through Microfold filter plates (V & P Scientific) and the filters counted. To account for unbound antibody retained in liquid on the filters, controls containing the same concentrations of antibody but no cells were done in parallel. Association constants and antigen copy number per target are calculated from the affinity test results and are reported in Table 7 below.

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